FIG. 5.

The v-cyclin.LacZ mutant establishes latency but fails to reactivate efficiently from C57Bl/6 mice. C57Bl/6 mice were infected i.p. with 10⁶ PFU of v-cyclin.LacZ (triangles), wt γHV68 (squares), or v-cyclin.MR (circles) virus, and cells were harvested 42 days postinfection for quantitation of the frequency of viral genome positive cells (A and B) and the frequency of cells reactivating virus (C and D). (A and B) Latently infected splenocytes or PECs were analyzed for the frequency of viral-genome-positive cells by limiting-dilution nested PCR. Ten PCRs were performed per cell dilution for each experiment, and PCR controls were run within each experiment. The numbers of individual experiments are indicated, and the standard errors of the means are shown (error bars). (C and D) Limiting-dilution ex vivo reactivation analyses using the same populations of splenocytes and PECs as for the experiments shown in panels A and B. Intact (live) cells were plated on MEF indicator monolayers to determine the frequency of ex vivo reactivation (closed symbols). In parallel, samples of each cell population were mechanically disrupted to measure preformed infectious virus (open symbols), which was demonstrated to be absent. Curve fit lines were derived by nonlinear-regression analysis, and symbols represent means and SEMs (error bars) of data from individual experiments as indicated. The dashed line is at 63%, the value which was used to calculate the frequency of genome-positive cells or the frequency of reactivating cells as indicated by a Poisson distribution. Data represent independent experiments as indicated, with each experiment containing cells pooled from four to seven mice. ∗∗∗, frequencies of reactivation for v-cyclin.LacZ and v-cyclin.MR were statistically different (PECs, P = 0.003; splenocytes, P = 0.043).